The DegP (HtrA) protease is essential for virulence in several Gram-negative pathogens: S. typhimurium, B. melitensis, Y. enterocolitica and P. aeruginosa. The phenotype of a degP knockout in Escherichia coli, temperature and oxidative sensitivity, suggests a role in virulence. The DegP protease is a multifunctional protein essential for the removal of misfolded and aggregated proteins in the periplasm of Gram-negative bacteria. We have identified the major pilin subunit of the Pap pilus, PapA, as a native DegP substrate and demonstrate vigorous proteolysis of this substrate in vitro. The DegP cleavage site in PapA was mapped and an in vitro cleavage assay suitable for HTS was developed. Hits that arise from the HTS will be passed through a series of secondary screening assays and in vivo models to identify bioavailable inhibitors of DegP that display good selectivity. We recently identified DegP homologues in S. pyogenes and S. aureus and demonstrated that a degP knockout in S. pyogenes has reduced virulence. We propose to validate S. aureus DegP as a virulence factor and develop this target for screening. There is ample precedent for the efficacy of protease inhibitors as therapeutic agents. Moreover, nonessential virulence targets may be less prone to resistance development.